Brain ischemia remains a disease without adequate treatment. Preventing ischemic brain injuries may be possible by preconditioning the brain with a brief period of hypoxia or with a volatile anesthetic to induce a state of tolerance to ischemia. The proposed studies will show that the concentration of calcium inside neurons ([Ca2+]i) is an integrating signal in these types of preconditioning. The role of Ca2* in preconditioning will be studied in 1) hypoxic preconditioning, which is produced by a brief period of non-injurious oxygen deprivation;2) anesthetic preconditioning, which follows exposure to the anesthetic isoflurane and 3) aging. in which preconditioning may be weak or of limited duration. These studies will involve organotypic hippocampal slice cultures, a powerful in vitro model of neuroprotection, ischemic tolerance and aging. We propose that successful preconditioning requires a moderate increase in [Ca2+]j to initiate a cascade of pro- survival, anti-apoptotic processes that include calmodulin, phosphorylation of survival kinases and the transcription factor CREB and the expression of neuroprotective genes. We will further define how this signaling cascade fades in the aging hippocampus, making older neurons less capable of benefiting from preconditioning. We will test 2 main hypotheses: 1) Moderate increases in [Ca2+]t are necessary for protective gene expression in both hypoxic and anesthetic preconditioning;and 2) Ca2+-dependent survival-signaling and gene expression associated with preconditioning fades with aging. The experimental plan involves measurements of [Ca2+]j with fluorescence microscopy, RNA interference and DNA microarrays to establish the importance of different elements of the Ca2+- survival cascade. Knowledge gained from these studies will define new ways of enhancing the brain's endogenous capacity to defend against injury from stroke. The work will also identify why the aging brain may not be able to benefit from the neuroprotective actions of anesthetics or other preconditioning treatments.